The long-term goal of our program is to develop safe and effective techniques to combat tissue damage caused by ischemia. The specific aims of this proposal are to investigate high-energy phosphate contents in human non-healing chronic wounds, to further improve our direct intracellular energy delivery technique, to use this technique in wound treatment, and to study some of the mechanisms by which intracellular energy supply enhances wound healing. Our central hypotheses are that depletion of high energy phosphate contents is the fundamental cause of non-healing chronic wounds; and that direct intracellular delivery of ATP to the wound will improve the microenvironment and substantially enhance the healing process. Results from our Phase I study also found that: 1) tissue biopsies taken from human chronic wounds showed significantly reduced high-energy phosphate contents; 2) intracellular ATP delivery using VitaSolTM substantially increased wound tissue high-energy phosphate contents in a rabbit ear model; and 3) intracellular ATP delivery caused extremely fast granular tissue growth. Using minimally invasive surgical procedures, we have also developed a new rabbit ischemic wound model that has been used successfully, for the first time, in diabetic animals. Results from the Phase I study have proven our basic concept. Three aims will be pursued in this Phase II study: 1. The first testable hypothesis is that wound tissue hypoxia results in decreased high-energy phosphate availability, and this reduction is the major cause of non-healing in chronic wounds. End point: high-energy phosphate contents in various chronic human wounds. 2. The second hypotheses to be tested is that unilamellar lipid vesicles containing encapsulated ATP can be manufactured on a large scale, and further, that these vesicles can be used for wound care with little systemic reactions. End point: a specific formulation of stable, lyophilized unilamellar lipid vesicles containing ATP with good tissue penetration suitable for wound care and a cGMP process capable of producing these vesicles. 3. The third testable hypothesis is that intracellular ATP delivery will increase wound tissue energy levels, induce coordinated upregulation of growth factors and other healing mechanisms to facilitate healing process. End point: tissue high-energy phosphate contents and their relationship with growth factors and healing speed. None of the 3 topics outlined above has been explored in the past. The success of this project and the expanded use of this new intracellular energy delivery technique will likely have a major impact on medicine. It will not only improve care for chronic wounds, but also improve our ability to treat various ischemic conditions, such as shock, stroke, severe trauma, heart attack, spinal cord injury, cardiopulmonary bypass, organ transplant, acute or chronic lung diseases, and many other conditions involving ischemia. The specific aims of this Phase II proposal are to investigate high-energy phosphate contents in human non-healing chronic wounds, to further improve our direct intracellular energy delivery technique, to use this technique in wound treatment, and to study some of the mechanisms by which intracellular energy supply enhances wound healing. Our Phase I project has improved formulation, encapsulation rate, and particle size of our intracellular ATP delivery preparation, measured high-energy phosphate contents in human chronic wounds, and used the intracellular ATP delivery in animal models. Our intracellular ATP delivery technique caused extremely fast granular tissue growth in rabbit ear wounds. None of the tasks outlined in the current project has been explored in the past. The success of this project and the expanded use of this new intracellular energy delivery technique will likely have a major impact on medicine. It will not only improve care for chronic wounds, but also improve our ability to treat various ischemic conditions, such as shock, stroke, severe trauma, heart attack, spinal cord injury, cardiopulmonary bypass, organ transplant, acute or chronic lung diseases, and many other conditions involving ischemia. [unreadable] [unreadable] [unreadable]